Raf-1 protein ser/thr kinase functions as a critical shuttle enzyme that connects stimulation of growth factor receptors and protein kinase C at the membrane with activation of early growth response genes in the nucleus. During the past year we have made considerable progress in identifying the upstream activators and downstream effectors of the Raf-1 signalling pathway. We have demonstrated that the effector domain of the activated Ras interacts with the amino-terminal regulatory domain of Raf-1. Whether this specific interaction of Ras with Raf-1 is sufficient for activation of Raf-1 kinase activity or requires additional co-factors remains to be elucidated. Thus far, attempts to activate Raf-1 in vitro with Ras-GTP have been unsuccessful. In an effort to understand the possible role of the co-factor in the Raf-1 activation mechanism, we have extended this study with cellular subfractions. One consequence of Ras- Raf binding is the activation of Raf/mitogen-activated protein kinase (MAP kinase) pathway. The activated Raf phosphorylates ser217 and ser221 of MAP kinase kinase (MEK), the only known physiological substrate of Raf-1. Dephosphorylation experiments together with phosphorylation mutant analysis demonstrated that phosphorylation of either residue is sufficient for maximal activation. Activated MEK phosphorylates the MAP kinase which, in turn, catalyzes the phosphorylation of several cytoplasmic proteins, including ribosomal protein S6 kinase and MAP kinase-activated protein kinase 1 and 2. In addition, activated MEK also catalyzes the phosphorylation of multiple oncogene class transcription factors leading to changes in gene transcription. In an effort to extend this study to the other family members of Raf (A-Raf and B-Raf), we have expressed wild-type and mutant versions of these proteins in Spodopera frugiperda (Sf9) cells and experiments are in progress.